The specific uptake of nutrients and extrusion of wast materials are fundamental properties of all metabolically active cells. In most cases which have been carefully examined, the transport of small, hydrophilic molecules through the hydrophobic membrane barrier is mediated by integral membrane proteins which show a high degree of specificity for their transport substrates. These "permeases" have been difficult to purify because of their hydrophobic nature and the lack of a convenient in vitro assay for transport. Recently, however, we have purified the D-mannitol specific Enzyme II of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) from Escherichia coli. This integral membrane permease catalyzes the concomitant vectorial transport and phosphorylation of D-mannitol in a process termed group translocation. Thus, it offers the distinct advantage of possessing an enzymic activity coupled to a transport function. This property, which is unusual among permeases, allows one to use classical enzymological methods to study the mechanism of transport. Specifically, we intend to investigate: The orientation of the mannitol Enzyme II polypeptide in the membrane using proteolytic enzymes, specific antibodies and chemical modification. The mechanism of the transport and phosphorylation reactions using inhibitors coupled with catalytic and binding studies. The amino acid sequence of the protein, particularly as this relates to its function and intramembrane topography. Successful completion of these studies will be an important step toward understanding the mechanism of transport in the PTS, a goal which has yet to be achieved for any transport system.